Conserved pathways from WASP/WAVE proteins to the Arp2/3 complex control actin dynamics in a huge range of cellular processes. In the previous cycle we learned how WASP is inhibited by the small molecule wiskostatin and integrates signals from the Cdc42 GTPase and kinases/phosphatases, and we developed a quantitative thermodynamic model for WASP autoinhibition. Here, we will focus on a new and important regulatory mechanism, in which the potency of WASP/WAVE proteins toward Arp2/3 complex is increased ~100-fold through oligomerization. This dramatic effect impacts on a wide range of WASP/WAVE signals, and we will explore it broadly in this proposal. WASP is activated by the EspFu protein from the human pathogen and potential bioterrorism agent, Enterohemorrhagic E. coli, during infection. We will determine the structure of a WASP-EspFu complex and use biochemical and biophysical methods to determine whether cooperativity between the multiple sequence repeats of EspFu results from WASP oligomerization. We will also use a multi-disciplinary approach to determine the relative importance of oligomerization and relief of autoinhibition during WASP activation by a battery of SH3-containing proteins. We have reconstituted the 400 kDa hetero-pentameric WAVE Regulatory Complex (WRC) and various subcomplexes from humans and flies, and will use these powerful reagents to learn how WAVE is controlled by upstream inputs including Rac and oligomerizing agents. These investigations will be complemented by structure determination of a key trimeric subcomplex of the WRC. The overall program will reveal the mechanisms by which diverse normal and disease-based signaling inputs control the activity of WASP and WAVE. Throughout, we will develop a novel concept that WASP/WAVE activity is controlled hierarchically;an inner layer of regulation governs the equilibrium between inactive and active states, and an outer layer governs the affinity of the active state for Arp2/3 complex. This concept will unify a large body of work in the field under a common mechanistic framework, explaining quantitatively how disparate signals converge on Arp2/3 complex to generate complex actin dynamics. The work will also reconcile two opposing models of WAVE regulation, and reveal common and distinct regulatory principles across the WASP/WAVE family. Our findings will address fundamental questions in biophysics, signal transduction and cell biology and could suggest new and improved methods for the detection and treatment of cancer, human genetic disorders and bacterial infection. Our research focuses on understanding the signaling pathways that connect Rho GTPases to WASP/WAVE proteins to the Arp2/3 complex. These pathways are critically involved in many normal biological processes and in numerous diseases, including metastatic cancer, immune disorders and bacterial/viral infection. An understanding of how the proteins communicate in these pathways, and can be diverted by bacteria, could lead to new agents for the diagnosis and treatment of many diseases.